Abstract
The central loop of G-quadruplex molecular beacons is a key element to sense target DNA or RNA sequences. In this study, circular dichroism spectroscopy (CD), thermal difference spectrum (TDS), non-denatured non-denaturing gel electrophoresis, and thermal stability analysis were used to investigate the effect of the central loop length on G-quadruplex features. Two series of G-quadruplexes, AG3TTAG3-(TTA)n-G3TTAG3T (n = 1–8) (named TTA series) and AG3TTTG3-(TTA)n-G3TTTG3T (n = 1–8) (named TTT series) were examined in K+ and Na+ solutions, respectively. CD and TDS spectral data indicated that TTA series adopted an antiparallel G-quadruplex structure in Na+ solution and a hybrid G-quadruplex structure in K+ solution respectively. TTT series exhibited a hybrid G-quadruplex structure in both Na+ and K+ solutions. UV melting curves indicated that the stability of G-quadruplex in both series was reduced by the elongation of central loop. Thermal stability analysis concluded that the G-quadruplex destabilization with long central loop is an entropy-driven process due to more flexible and longer central loops.
Highlights
Many guanine-rich sequences in human genome can fold into G-quadruplex (G4) structure.The G4 consists of stacked G-quartets, each of which consists of four guanine residues connecting via Hoogsteen hydrogen bonds in a plane (Figure 1a)
As the central loop length becomes increased, the negative peak at 262 nm shows a stepwise leftward shift slightly, the positive peak at 295 nm remains unchanged, and the positive peak at 243 nm becomes diminished gradually. The shape of these circular dichroism spectroscopy (CD) spectra and the elliptic signals change very slightly. These results suggest clearly that the the central loop increases from (TTA) series fold into an antiparallel G4 structure in Na+ solution, the central loop elongation only induces a minor local conformational change without changing the overall G4 topology
CD and thermal difference spectrum (TDS) results have concluded that the TTA series in Na+ solution adopt the intramolecular antiparallel G4 structure, and the central loop elongation causes a minor change of elliptic signal without changing the overall G4 topology
Summary
Many guanine-rich sequences in human genome can fold into G-quadruplex (G4) structure.The G4 consists of stacked G-quartets ( named G-tetrads), each of which consists of four guanine residues connecting via Hoogsteen hydrogen bonds in a plane (Figure 1a). The G-quartets in G4 structure stack together linked with two side loops and one central loop. According to the strand polarities, the G4 structures are classified into a parallel, antiparallel or hybrid parallel/antiparallel folding topology (Figure 1b–f). These highly ordered G4 structures are found in telomeres [1,2,3], promoters [4,5], and 50 -UTR regions of mRNAs [6,7] of eukaryotic systems, suggesting their biological significance on gene expression [8,9,10,11,12]. Structure stability of G4 is usually influenced by the G-tract number [13,14,15], the loop of eukaryotic sequence [16,17,18,19,20,21,22], the loop size [10,13,14,16,17,18,19,20,21,22,23,24,25,26,27,28], and metal ions [29,30,31]
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